The present disclosure relates to a microscopy system, a microscopy method, and a computer readable recording medium.
In observation of a subject having a height difference by use of an industrial microscope, or in observation of a subject having a thickness, such as a cell nucleus or a stem cell, by use of a biological microscope having a focal depth of several ten micrometers; there is a user need for quick identification of a region of interest present in a depth direction (Z direction) along the optical axis of the observation optical system. For this need, there is a method, in which plural images having different focal planes are acquired by sequential imaging being performed while the focal plane of the observation optical system is shifted along the optical axis, and these plural images are displayed by being arranged three-dimensionally. By this three-dimensional display, three-dimensional information is able to be observed from an arbitrary direction, and the position of a region of interest is able to be checked. The plural images with the different focal planes acquired as described above are collectively called Z-stack images.
However, when plural focused structures are present in the depth direction like in a transparent subject or a subject having a height difference, by the conventionally three-dimensional display, a state, in which another structure present more shallowly in the observation direction or a blur due to an out-of-focus position hides a structure that is in the background (occlusion), is caused, and thus there is a region where its structure is unable to be directly (intuitively) seen. When occlusion is caused, an image, which has been captured at a position where a structure desired to be observed is present, needs to be searched for and checked two-dimensionally. There is also a method, in which a three-dimensional position is checked by display of only a structure having the maximum luminance in the depth direction, but this method has a problem that structures not having the maximum luminance are not reproduced.
Therefore, a method, in which a search is performed in the depth direction after presence and X-Y positions of two-dimensionally overlapping structures are checked by generation of an all-focused image from Z-stack images, may be used. Methods of generating an all-focused image include: a method of reconstructing a multi-focused image synthesized by superimposition of Z-stack images; and a method of extracting a focused area in each of Z-stack images and synthesizing the focused areas. Such an all-focused image is useful for screening of plural structural arrangements present in the depth direction.
For example, in Japanese Patent Application Laid-open No. 2014-021490, a method, in which an area desired to be observed is selected by use of a user interface, from an all-focused image generated from Z-stack images, and Z-stack images that are in focus are extracted and displayed based on in-focus-ness, is disclosed.
Further, in Japanese Patent Application Laid-open No. 2014-021489, a method, in which in-focus-ness is calculated in Z-stack images, extraction candidates are selected based on in-focus-ness in the depth direction, weighting according to the in-focus-ness is executed, and synthesis is performed, is disclosed. According to Japanese Patent Application Laid-open No. 2014-021489, a depth map is able to be generated based on a peak of in-focus-ness at each X-Y position, and a Z-position of a peak of in-focus-ness is able to be known from this depth map.
Further, in International Publication WO No. 2011/158498, a technique, in which two images respectively focused at a near end side and a far end side of a subject, and an all-focused image generated by imaging being executed while an image sensor is swept from the near end side to the far end side of the subject, are acquired, the images respectively focused at the near end side and the far end side are reconstructed from the all-focused image, thereby an amount of blurring in a partial area in an image is calculated, and thereby a distance from the optical system to the subject is acquired and a distance map is generated, is disclosed.